Lactic acid bacteria (LAB) are of considerable importance in winemaking. The so-called malolactic fermentation (MLF), the conversion of L-malic acid to L-lactic acid and CO.sub.2 by certain strains of lactic acid bacteria contributes much to the final wine quality. The conversion of malic acid to lactic acid reduces the acidity of wine. This is desirable in wines from cool wine growing areas which tend to have high acid contents (Wibowo et al., 1985, Am. J. Enol. Vitic. 36:302-313; Henick-Kling, 1988, in "Modern Method of Plant Analysis" New Serial Vol. 6, Springer Verlag, Berlin, p. 276-316; Radler, 1966, Zentralbl. Bakteriol. Parasitenk. Intektionskr. Hyg. Abt 2. 120:237-287; Amerine and Kunkee, 1968, Ann. Rev. Microbiol. 22:323-358; Ribereau-Gayon and Peynaud, 1975, Traite d'Oenolgie Tome 2, Dunod, Paris).
Typically, spontaneous MLF occurs in wines of pH above 3.4 and is much delayed and irregular in wines of pH below 3.4 where MLF is most desirable for deacidification. This is due to the strong inhibition of growth of LAB at low pH. Inoculation of such wines with a large number of viable bacteria avoids the necessary growth before MLF is carried out. With a suitably prepared starter culture such as obtained with the medium described here, a wine can be inoculated with a cell density of 5.times.10.sup.6 and 5.times.10.sup.7 viable bacteria/ml. This represents a stationary culture at maximum cell density in wine, very little or no further growth is necessary for complete conversion of the malic acid. This procedure of inoculating with stationary phase cultures also is effective in avoiding phage interference (Henick-Kling et al., J. Appl. Bacterial. 61:525-534; 1986). It has been shown that Leuconostoc oenos can be attacked by bacteriophage during growth in wine and infection and lysis inhibits MLF (Sozzi et al., Rev. Suisse Vitic. Arboric. Hortic. 14:17-23; 1982. Henick-Kling et al., J. Appl. Bacteriol. 61:287-293; 1986). Recent investigations into the presence of phages in L. oenos show that 50% of all strains isolated from wine contain temporary phages. This and previous demonstrations of the presence of phages in wine show that cultures of L. oenos are susceptible to phage-induced lysis during growth in wine. Inoculation with a high cell density allows to avoid necessary growth and possible phage interference. Further, inoculation with high cell density (5.times.10.sup.6 to 5.times.10.sup.7 viable bacteria/ml) ensures dominance of the introduced culture over indigenous LAB such as undesirable strains of Pediococcus. Inoculation with a dominating, selected strain of LAB gives protection against undesirable strains of Pediococcus and Lactobacillus. Thus the winemaker can control which strain of bacteria carries out MLF and he can give the wine the desired flavor characteristic of the selected strain. Stationary cell cultures may also be used in a cell reactor system (Gestrelius et al., U.S. Pat. No. 4,380,552). Also, wines which did undergo MLF are considered to be biologically stable (Kunkee, 1974, in "The Yeasts" Vol. 3, Yeast Technology, ACS, Wash. D.C.).
In winemaking, traditionally, the development of the indigenous bacterial flora, which originates from grapes and winery equipment, has been stimulated by several means: the use of none or small amounts of SO.sub.2, adjustment of the pH to 3.2 or higher, maintaining a temperature of 16-20.degree. C., and extended contact with yeast lees. More recently, starter cultures composed of lactic acid bacteria have been used to induce MLF (King, 1986, Dev. Ind. Microbiol. 26:311-321; Mayer et al., 1983, Schweiz. S. Obstund Weinball. 119:197-200; 1984, ibid 120:191-193; Lafon-Lafourcade, 1983, in "Biotechnology", Vol. 5, Verlag Chemie, Weinheim p. 43-53; Champagne et al., 1989, Appl. Environ. Microbiol. 55:2488-2492).
There are three genera of lactic acid bacteria, that are generally associated with MLF. They are species of Lactobacillus, Pediococcus, and Leuconostoc oenos (Radler, 1966, supra; Kunkee, 1967, Adv. Appl. Microbiol. 9:235-279; Maret et al., 1977, Ann. Technol. Agricoles 26:235-273; 1979, ibid 28:41-55). Leuconostoc oenos is generally the most predominant genus during active MLF, since this species is highly tolerant to the high acidity and ethanol in wine (Maret et al., 1979, supra; Davis et al., 1986, Appl. Environ. Microbiol. 51:539-545).
Strains of Leuconostoc oenos are the preferred lactic acid bacteria to carry out malolactic fermentation (MLF) because of their tolerance to low pH and alcohol and because of their preferred flavors produced.
Selected strains of LAB for induction of MLF in wine may comprise strains of Leuconostoc oenos, Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus brevis, and Lactobacillus casei. Other options of reducing the content of malic acid in wine by the yeasts Schizosaccharomyces pombe or Schizosaccharomyces maledivorans have not given satisfactory wine quality.
Various studies have been performed to select lactic acid bacteria strains which are highly tolerant to the low pH and the alcohol content of wine (Kunkee, 1967, supra; Kunkee et al., 1964, Am. J. Enol. Vitic. 15:178-183; Beelman et al., 1977, Am. J. Enol Vitic. 28:159-165; 1980, ibid 31:269-276; 1982, Dev. Ind. Microbiol. 23:107-121; Silver et al., 1981, Am. J. Enol Vitic. 32:64-72; Lafon-Lafourcade, 1983, supra; Sandine, 1979, Pfizer Cheese Monographs, Vol VI, Pfizer Inc., NY; Sandine et al., 1985, U.S. Pat. No. 4,547,373; King 1985, U.S. Pat. No. 4,562,077; 1987, EPO 141 878 B1). Some strains highly active in wine at low pH have been patented (Sandine et al., 1985, U.S. Pat. No. 4,547,373; King, 1985, U.S. Pat. No. 4,562,077). Very few studies have been performed on the biomass production of malolactic starter cultures for use in wine (Champagne et al., 1989, Appl. Environ, Microbiol. 55:2488-2492; and Lebensm. - Wiss. u. -Technol. 22:376-381; Schneider et al., 1987, FEMS, Microbiology Reviews 46/3: P. P56 D7). Previously described media for the production of Leuconostoc oenos biomass are based on grape juice, apple juice, or pear juice (Davis et al., 1985, supra) which is generally diluted and supplemented with yeast extract and tomato juice, Tween 80 and peptone or a mixture of vitamins (Krieger, 1989, Optimierung des biologischen Saureabbaus in Wein mit Starterkylturen., Ph.D. Thesis, Universitat Hohenheim, FRG). Recently, Champagne et al. reported the production of Leuconostoc oenos in apple juice based media (1989, Appl. Environ. Microbiol. 55:2488-2492) as well as the production of Leuconostoc biomass under pH control.